This application aims to understand the mechanisms and pathways that the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa use to counter oxidative stress produced by host immune response. Staphylococcus aureus is a human pathogen responsible for most wound and hospital-acquired infections and is responsible for the highest mortality of any infectious diseases in the United States. The extensive use of antibiotics to treat S. aureus infections has led to the emergence and rapid spread of highly virulent, drug-resistant S. aureus strains (MRSA) worldwide. Virulence suppression provides an alternative strategy to effectively reduce pathogenic potential without asserting selective pressure for developing resistances. My laboratory has identified new global regulatory proteins that control the virulence of S. aureus. Mechanistic studies indicate that these proteins possess redox-active Cys residues to sense reactive oxygen species (ROS). Human innate immune response produces high concentrations of ROS to counter bacterial infections. Therefore, our discovery suits well that a pathogen inside a human host uses oxidation-sensing mechanisms to sense the host immune response and regulate a global change of its properties including virulence and metabolisms. We employ an advanced chemical proteomic technology, based on activity-based protein profiling (ABPP) methods, to quantitatively profile functional cysteines to profile oxidation-sensitive cysteines in the proteomes of S. aureus and P. aeruginosa. Besides identifying additional regulatory proteins that are redox active, we also aim to discover major metabolic pathways that are dramatically affected by ROS treatment. A global view of how these pathogens respond to oxidative stress will emerge from the proposed program of studies with key virulence determinants identified and characterized. Other than Cys-oxidation, we have made a surprising new discovery that the functional Cys residues in many of these proteins that are oxidation sensitive can also be phosphorylated. The Cys-phosphorylated form of the regulatory protein correlates to the low virulence state of the pathogen just like the Cys-oxidized form of these regulators. Thus, these master switches possess multiple regulatory mechanisms to sense and respond to diverse host and/or environmental signals including oxidative stress. The discovery that Cys-phosphorylation plays important roles in biological regulation is unprecedented and is itself fundamentally interesting. The underlying mechanism and pathways will be elucidated.